This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pyruvate kinase is an essential glycolytic enzyme whose activity is highly regulated. There are 4 isoforms of the enzyme. The R and L isoforms are specific for red blood cells and liver respectively. The other two isoforms, M1 and M2, are splice variant of the same gene with only one exon difference. While the M2 isoform is highly expressed in embryonic cells and adult stem cells, most adult tissues express the M1 isoform. However, all cancer cells are found to switch back completely M2 expression. Although the cause of this switch is currently unknown, this differential expression between normal tissues and tumor makes the M2 isoform a drug target for cancer treatment. We have found several compounds that specifically inhibit the M2 isoform and not the M1 isoform. Since the lead compounds still have fairly high IC50, we would like to determine the structure of M2 bound to the inhibitors so rational modification can be made to generate better compounds. In addition, in contrast to PKM1 or the L isoforms, PKM2 preferentially interacts with phosphotyrosine peptide libraries. From mutagenesis studies, we also found that this interaction reduces its enzyme activity, presumably by displacing the allosteric activator FBP. This may explain why cancer cells have PKM2 isoform, since the extra level of control is advantageous for cancer cells to respond to deprivation of nutrients. In order to understand the regulation of PKM2 activity better, we would like to see at the atomic level, how the phosphopeptide competes with FBP.